Noradrenergic stimulation of α1 adrenoceptors in the medial prefrontal cortex mediates acute stress-induced facilitation of seizures in mice

Stress is one of the critical facilitators for seizure induction in patients with epilepsy. However, the neural mechanisms underlying this facilitation remain poorly understood. Here, we investigated whether noradrenaline (NA) transmission enhanced by stress exposure facilitates the induction of medial prefrontal cortex (mPFC)-originated seizures. In mPFC slices, whole-cell current-clamp recordings revealed that bath application of picrotoxin induced sporadic epileptiform activities (EAs), which consisted of depolarization with bursts of action potentials in layer 5 pyramidal cells. Addition of NA dramatically shortened the latency and increased the number of EAs. Simultaneous whole-cell and field potential recordings revealed that the EAs are synchronous in the mPFC local circuit. Terazosin, but not atipamezole or timolol, inhibited EA facilitation, indicating the involvement of α1 adrenoceptors. Intra-mPFC picrotoxin infusion induced seizures in mice in vivo. Addition of NA substantially shortened the seizure latency, while co-infusion of terazosin into the mPFC inhibited the effect of NA. Finally, acute restraint stress shortened the latency of intra-mPFC picrotoxin infusion-induced seizures, whereas prior infusion of terazosin reversed this stress-induced shortening of seizure latency. Our findings suggest that stress facilitates the induction of mPFC-originated seizures via NA stimulation of α1 adrenoceptors.

For recording evoked inhibitory postsynaptic currents (eIPSCs) using the whole-cell voltage-clamp technique, the membrane potentials were held at + 10 mV in the presence of a glutamate receptor antagonist KYNA (2 mM), and electrical stimulations were applied as cathodal square-wave pulses of 200-μs duration with an intensity of up to 30 μA using a glass electrode that was located in layer 2/3 of the mPFC and filled with normal Ringer's solution. The eIPSC amplitudes were measured in periods of 0-30 s before the end of the drug applications. The picrotoxin effects were evaluated by comparing the average values of these periods.
For whole-cell current-clamp recordings, membrane potentials were recorded without holding current injection. The generation of EAs, defined as prolonged depolarization accompanied with three or more burst firings, was recorded for 15 min (time 0 = start of drug application). EA latency was measured as the time of the induction of the first EA after drug application. If EA was not induced within 15 min, the latency was calculated as 15 min. The duration and number of burst firings of an EA were measured on the first EA in periods of 0-5 min and 10-15 min after drug application (picrotoxin, picrotoxin + NA, or picrotoxin + phenylephrine). In pharmacological experiments, EA frequency was measured in periods of 0-3 min before and 2-5 min after the application of antagonists. In some experiments, whole-cell current-clamp and field potential recordings were obtained simultaneously. The field potential recordings were obtained using a glass electrode placed in the mPFC L5 and containing 2.5 M NaCl with a resistance of 1-3 MΩ in Ringer's solution.
Data were amplified with a Multiclamp 700B (Molecular Devices, Foster City, CA, USA) and low-pass-filtered at 3 kHz. Additionally, field potential data were high-pass-filtered at 0.1 Hz. Voltage-clamp, current-clamp, and simultaneous recording data were then digitized at 20, 6, and 10 kHz with an A/D interface (Digidata 1440A; Molecular Devices), respectively, and stored on a computer using a Clampfit 10.5 (Molecular Devices). Field potential data were further low-pass-filtered at 30 Hz with the Clampfit 10.5. The correlation between EAs and field potentials was evaluated by cross-correlation analysis. The cross-correlogram was constructed using onset time points of the first peaks of EAs and the peaks of negative shifts of field potentials. The calculation was based www.nature.com/scientificreports/ on the previously described methods 35 and performed using the 'scipy.signal.correlate' function in the SciPy library in Python. A bin width of 300 ms was used.
Histochemistry. Histochemical examinations of recorded neurons were implemented as described previously 26,33,34 . After the recordings, the slices were fixed overnight in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4 °C. After three rinses with 0.05 M PBS, the slices were then incubated in 0.6% H 2 O 2 in methanol for 30 min at room temperature to eliminate endogenous peroxidase activity, followed by 3 h of incubation in an avidin-biotin-peroxidase complex using a R.T.U. ABC Reagent (Vector laboratories, Burlingame, CA, USA) diluted fourfold in PBS containing 0.3% Triton X-100. The slices were then rinsed in 0.05 M Tris-HCl (pH 7.5) and incubated in 0.05% 3,3′-diaminobenzidine (DAB; Nacalai Tesque, Kyoto, Japan) solution. Next, the biocytinfilled cells were stained brown, and the reaction was stopped by rinsing with 0.05 M PBS. The biocytin-labeled cells were identified under a microscope (BZ-9000; Keyence, Osaka, Japan). L5 pyramidal cells were identified according to the following characteristics: the depth of the cells from the midline (350-550 μm) and shapes of the soma and apical dendrites.
Surgery and intra-mPFC infusion. Intra-mPFC infusions were performed as previously described with slight modification [36][37][38] . Mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and implanted with 25-gauge stainless-steel guide cannulae (o.d., 0.51 mm; i.d., 0.26 mm) above the mPFC (1.8 mm rostral, ± 1.3 mm lateral, − 1.5 mm ventral to bregma, at a 20° angle from the vertical axis in the mediolateral plane; bilateral) 39 . After surgery, the mice were housed individually and allowed to recover for at least 5 days. For intra-mPFC infusions, 33-gauge stainless-steel infusion cannulae (o.d., 0.2 mm; i.d., 0.08 mm) were inserted bilaterally into the guide cannula, with the infusion cannulae protruded 1.1 mm from the tip of the guide cannula to reach the mPFC. The drugs (or vehicle) were administered bilaterally in a volume of 0.2 μL/side and at a rate of 0.2 μL/min. After the infusion, the infusion cannulae were kept in place for an additional 1 min after intra-mPFC infusion to prevent backflow.
Behavioral seizure observation. All mice were handled for 2 consecutive days, followed by habituation to a transparent testing chamber (32 cm × 22 cm × 13.5 cm; Natsume Seisakusho, Tokyo, Japan) for 30 min. On day 1, each mouse was administered a bilateral injection of picrotoxin (0.1 nmol/side) into the mPFC, placed in the testing chamber for 1800s (30 min), and videotaped. On day 2, the effects of intra-mPFC infusion of NA were tested in each mouse by administering a bilateral injection of either picrotoxin (0.1 nmol/side) only, picrotoxin + terazosin (picrotoxin: 0.1 nmol/side, terazosin: 7.5 nmol/side), picrotoxin + NA (picrotoxin: 0.1 nmol/ side, NA: 10 nmol/side), or picrotoxin + NA + terazosin (picrotoxin: 0.1 nmol/side, NA: 10 nmol/side, terazosin: 7.5 nmol/side) into the mPFC. The mice were then immediately placed in the testing chamber for 1800s and videotaped. On day 2, the effects of acute restraint stress were tested in each mouse by administering a bilateral injection of either vehicle (PBS) or terazosin (7.5 nmol/side) into the mPFC, followed by 20 min immobilization using a plastic bag (DecapiCones; Braintree Scientific, Braintree, MA, USA). Immediately after the restraint stress, each mouse was administered a bilateral injection of picrotoxin (0.1 nmol/side) into the mPFC, placed in the testing chamber for 1800s, and videotaped. Behavioral seizures were classified on the basis of the modified Racine scale 40,41 : stage 1, absence-like immobility; stage 2, hunching with facial or manual automatisms; stage 3, rearing with facial or manual automatisms and forelimb clonus; stage 4, repeated rearing with continuous forelimb clonus and falling; and stage 5, generalized tonic-clonic convulsions with lateral recumbence or jumping and wild running followed by generalized convulsions. Stage 1 and 2 were reported as non-convulsive seizures with no clear motor component, whereas stage 3 and above were convulsive motor seizures 41 . Thus, we measured the latency of stage 3 seizures.
Histology. After the behavioral tests, histological analyses were performed to confirm the infusion sites in the mPFC using the methods reported in our previous studies 26,27,[36][37][38] . Briefly, the mice were decapitated, and the brains were rapidly dissected and frozen in powdered dry ice. Coronal Sects. (50 μm thick) were prepared on a cryostat, thaw-mounted onto slides, stained with thionin, and examined under a microscope (BZ-9000). Data from mice with incorrect infusion placements (n = 22) were excluded from statistical analyses.
Statistical analyses. Data are expressed as mean ± standard error of the mean. The data were compared using Student's t-test or paired t-test when comparing two groups, one-way repeated measures ANOVA with post hoc Holm-Sidak's multiple comparison test or two-way repeated measures ANOVA with post hoc Bonferroni's multiple comparison test when comparing more than two groups. All analyses were performed using Prism 6 (GraphPad Software, La Jolla, CA, USA). We also confirmed that all experiments conducted in this study had adequate statistical power (greater than 0.9) using G*Power software 3.1 42 . Statistical significance was set at P value < 0.05.

NA promotes induction of EAs in mPFC L5 pyramidal cells under the suppression of GABA A receptor-mediated inhibition in vitro.
We first examined the effects of picrotoxin, a GABA A receptor antagonist, on eIPSCs in mPFC L5 pyramidal cells using in vitro whole-cell current clamp recordings (Fig. 1a).  www.nature.com/scientificreports/ P = 0.0034; and control vs. 30 µM, P = 0.0035, n = 5, one-way repeated measures ANOVA with post hoc Holm-Sidak's test, Fig. 1b,c). Thus, we used 30 µM picrotoxin to reproduce an epileptiform state 5,43 in the following experiments. Bath application of picrotoxin induced an EA, which consisted of a prolonged depolarization, the so-called paroxysmal depolarization shift (PDS) 44 , accompanied with more than 3 burst firings in 1 of 6 cells tested (Fig. 1d,e). The first EA was observed at least 7 min after the application of picrotoxin. The number of EAs generated during the 15 min recording (time 0 = start of picrotoxin application) was 1. EA was not induced during the 15 min recording in the remaining 5 cells. Picrotoxin did not significantly affect resting membrane potentials (control, − 73.15 ± 1.06 mV vs. picrotoxin, − 73.06 ± 1.10 mV, n = 7, t 6 = 0.3192, P = 0.7604, paired t-test). These findings indicate that picrotoxin application alone induces EA quite sporadically.

EAs are induced synchronously via glutamatergic transmission in the mPFC local circuit.
Using simultaneous whole-cell current-clamp and field potential recordings, we next investigated whether EAs are synchronous. Clear population activities were not observed shortly after the application of picrotoxin + NA, which is the time at which small EAs were generated as described above. However, several minutes later, gradual augmentation of population activities and emergence of clear negative potentials, which were time-locked to enlarged EAs, were observed (n = 6, Fig. 2a,b,c). To investigate the temporal relationship between the onset times of EAs and negative shifts in the field potentials more clearly, we conducted cross-correlation analysis. The highest peak was detected at time 0 with a bin width of 300 ms (Fig. 2d), indicating that EAs and negative shifts of field potentials occurred within 300 ms. These results indicate that EAs are synchronous in the mPFC.
We next addressed whether EAs are induced in the mPFC local circuits using mPFC-isolated slices (Fig. 3a). Whole-cell current-clamp recordings revealed that EAs were still induced in these slices after picrotoxin + NA application (6 of 6 cells; Fig. 3b), indicating that mPFC local circuits are sufficient for inducing EAs.

Restraint stress promotes the induction of seizures via α 1 adrenoceptors in the mPFC. Because
previous studies demonstrated that NA levels in the mPFC increased during restraint stress exposure 14-17 , we finally examined whether acute restraint stress promotes the induction of seizures via α 1 adrenoceptor stimulation in the mPFC. Mice received bilateral intra-mPFC infusion of picrotoxin on day 1. On day 2, the mice were randomly divided into two groups. One group received intra-mPFC infusion of vehicle, followed by a 20-min restraint stress and then intra-mPFC picrotoxin infusion (Fig. 7a). The second group received similar treatment, with the exception of intra-mPFC infusion of terazosin instead of vehicle (Fig. 7a). Two-way repeated measures ANOVA revealed a significant interaction (F 1, 12 = 9.851, P = 0.0086, Fig. 7b). The latency of seizure induction in the picrotoxin + restraint stress + vehicle group on day 2 was significantly shorter than that on day 1 (day 1,   Fig. 7b). The latency of seizures in the picrotoxin + restraint stress + terazosin group was not significantly different between day 1 and 2 (day 1, 15.70 ± 2.75 min vs. day 2, 20.67 ± 3.79 min, P = 0.187, n = 7, post hoc Bonferroni's test, Fig. 7b). However, it should be noted that the shortened latency induced by restraint stress was reversed by intra-mPFC infusion of terazosin on day 2 (picrotoxin + restraint stress + vehicle, 10.84 ± 1.00 min, n = 7 vs. picrotoxin + restraint stress + terazosin, 20.67 ± 3.79 min, n = 7, P = 0.0276, post hoc Bonferroni's test, Fig. 7b). These results indicate that restraint stress promotes the induction of mPFC-originated seizures through the activation of α 1 adrenoceptors in the mPFC.

Discussion
The main findings of the present study were as follows: (1) bath application of NA promotes EA induction in mPFC L5 pyramidal cells via α 1 but not α 2 or β adrenoceptors; (2) NA-facilitated EAs are synchronously induced in the mPFC local circuit; (3) latency of intra-mPFC picrotoxin-induced seizures are shortened by co-infusion of NA, which was recovered by α 1 adrenoceptor antagonism; and (4) acute restraint stress exposure shortens the latency of intra-mPFC picrotoxin-induced seizures via α 1 adrenoceptors. All these findings suggest that the activation of α 1 adrenoceptors in the mPFC by NA may contribute to the generation of stress-induced seizures in patients with epilepsy. Electrophysiological recordings revealed that NA promotes EA induction in mPFC L5 pyramidal cells via α 1 adrenoceptor stimulation. This result is in contrast to the finding that α 1 adrenoceptor stimulation suppressed EA in hippocampal slices 45 . Alpha 1 adrenoceptors are expressed in not only glutamatergic pyramidal cells but also GABAergic interneurons of the mPFC 46 . The stimulation of α 1 adrenoceptors depolarizes GABAergic interneurons, leading to an enhanced GABAergic transmission onto mPFC pyramidal cells 47 . Considering that GABA A receptors were inhibited by picrotoxin in our experimental condition, we did not observe the effect of augmented GABAergic transmission caused by α 1 adrenoceptor stimulation. However, we have previously reported that bath application of NA induces depolarization and increases EPSCs in mPFC L5 pyramidal cells in normal ACSF 26 , indicating that α 1 adrenoceptor stimulation in the mPFC local circuit may have an excitatory net effect even if GABAergic transmission was augmented by NA. In the hippocampal CA1, the stimulation of postsynaptic α 1A adrenoceptors increases firing activity of GABAergic interneurons, leading to an increased IPSC frequency in pyramidal neurons 45 . This effect of NA may suppress TLE seizures 45 . It has been reported that NA also facilitates IPSCs via stimulating α 1A adrenoceptors expressed at presynaptic terminals of GABAergic neurons in the basolateral amygdala 48 . However, this effect of NA is impaired by chronic stress exposure, suggesting that stress may potentiate the induction of seizures originated from the basolateral amygdala 48 . Thus, these findings suggest that the effects of α 1 adrenoceptor stimulation on local network activity depend on the brain regions and physiological/pathophysiological conditions. Nevertheless, in the absence of GABAergic function in FLE patients, the stimulation of α 1 adrenoceptors may facilitate hyperexcitability in the mPFC local circuit. www.nature.com/scientificreports/ Glutamatergic transmission has been reported to be crucial for inducing EAs [49][50][51] . Consistent with this, our pharmacological examinations revealed that CNQX as well as AP-5 suppressed EAs in the mPFC slices, indicating the critical role of both AMPA and NMDA receptors in EA induction. Although NMDA receptors are blocked by Mg 2+ at resting membrane potential, this block is removed when the membrane is depolarized. Because NA induces depolarization in mPFC L5 pyramidal cells 26 , NMDA receptor-mediated excitation accompanied with AMPA receptor-mediated transmission may contribute to the facilitation of EA induction. This is supported by a previous finding showing that the hyperactivation of NMDA receptors with free extracellular Mg 2+ induces EA in rat hippocampal slices 52 .
Simultaneous whole-cell and field potential recordings revealed that individual EAs caused by bath application of picrotoxin + NA were time-locked to individual large field potential changes, which corresponded to population spikes, indicating the synchronism of the EAs. Additionally, EAs were observed in the mPFC-isolated slices that did not include other brain regions, suggesting that mPFC local circuitry is sufficient to induce EA. Because the mPFC contains recurrent excitatory connections 53 , activation of pyramidal cells by NA could trigger synchronous neuronal activities under the suppression of GABA A receptor-mediated inhibition.
Systemic administration of convulsants, such as picrotoxin, pentylenetetrazol, pilocarpine, and kainic acid (KA), has frequently been used to generate animal models of seizures [54][55][56][57] . Additionally, the local infusion of KA into the hippocampus and amygdala, which are two major focal seizure sites, is used to generate TLE models 58,59 . However, there are few studies involving FLE models. A previous report showed that repeated intra-PFC infusion of bicuculline, a competitive GABA A receptor antagonist, in adolescent rats induced focal EAs 60 . In contrast, the present study demonstrated that single intra-mPFC infusion of picrotoxin induced stage 3 seizures in most animals examined (39 out of 49 mice). However, it should be noted that we might have missed the occurrence of stages 1-2 seizures because of the paucity of observable behavior changes in stages 1-2 of the Racine scale 40,41 . Thus, single intra-mPFC picrotoxin infusion-induced seizures could be a simple and useful model of FLE. Although the local circuit activity is a primarily important for the mPFC-originated seizures, other brain regions might also be involved in seizure induction. The mPFC is known to project to and receive excitatory inputs from the thalamus that is one of the critical brain regions associated with epilepsy 61 . Thus, it is likely that abnormal excitability generated in the mPFC via stress exposure might be transmitted to this brain region, leading to the generation of epileptic seizures.
We found that the latency of stage 3 seizures induced by intra-mPFC picrotoxin + NA infusion was considerably shorter than that induced by intra-mPFC picrotoxin infusion alone. Furthermore, this shortened latency was reversed by intra-mPFC co-infusion of terazosin, indicating the involvement of α 1 adrenoceptor stimulation in the shortening of the latency. Considering the in vitro electrophysiological data, these effects of NA and terazosin infusion on seizure latency may be attributed to the facilitation of EA generation and EA suppression by NA and terazosin, respectively, in the mPFC.
Previous studies have demonstrated the involvement of α 1 adrenoceptors in pro-and anti-convulsant effects of NA. For example, transgenic mice with overexpression of α 1B adrenoceptors exhibited increased seizures 62 , whereas α 1A adrenoceptor agonist inhibited seizures in rat models 63 . Accordingly, it is predicted that α 1B adrenoceptors might have played a critical role for facilitating the seizure induction observed in the present study. However, since the function and expression of each α 1 adrenoceptor subtype may be different in distinct brain regions 45,48 , further studies are required to elucidate the α 1 adrenoceptor subtype(s) that are critical for facilitating the induction of mPFC-originated seizures.
Previous studies have reported that stress induces seizures [6][7][8][9] . Consistent with these findings, we demonstrated that restraint stress shortened the latency of seizure induction caused by intra-mPFC picrotoxin infusion. To our knowledge, this is the first study demonstrating that single acute restraint stress exposure affects the induction of mPFC-originated seizures. However, one study has reported that repeated restraint stress increased seizure susceptibility via the hippocampus 64 . Stress activates LC neurons, leading to increased NA release in seizure foci, such as the mPFC, hippocampus, and amygdala [14][15][16][17][18][19] . Although we did not measure the level of NA in the mPFC during the restraint stress exposure in the present study, previous studies in rats demonstrated that NA levels in the mPFC increased to approximately 150% from baseline levels during restraint stress and this increase persisted for about 30 min [14][15][16][17] , suggesting the critical role of NA for seizure facilitation. Consistent with this, we found that intra-mPFC terazosin infusion before stress exposure suppressed the latency shortening effect of stress. All these in vivo behavioral data combined with the in vitro electrophysiological results strongly suggest that stress facilitates seizure induction via elevated NA, which stimulates α 1 adrenoceptors in the mPFC. It should be noted that restraint stress has been reported to increase the levels of not only NA but also dopamine and serotonin 17,65 . Thus, the possible involvement of these neurotransmitters in stress-induced seizure facilitation should be examined in future studies.
The mPFC can be divided into the prelimbic and infralimbic subregions. Because of a technical limitation, we could not selectively infuse drugs into each of the mPFC subregions. Further studies may be necessary to determine the role of each subregion on the stress-facilitated seizure induction.
In conclusion, our study demonstrates that NA released in the mPFC may contribute to stress-induced seizures via α 1 adrenoceptor stimulation. Thus, α 1 adrenoceptor blockers could be used as prophylactic and therapeutic drugs for stress-induced seizures in patients with focal mPFC epilepsy.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.